World-wide survey of an Accord insertion and its association with DDT resistance in Drosophila melanogaster

Previous work showed that insecticide resistance in Drosophila melanogaster is correlated with the insertion of an Accord-like element into the 5' region of the cytochrome P450 gene, Cyp6g1. Here, we study the distribution of the Accord-like element in 673 recently collected D. melanogaster lines from 34 world-wide populations. We also examine the extent of microsatellite variability along a 180-kilobase (kb) genomic region of chromosome II encompassing the resistance gene. We confirm a 100% correlation of the Accord insertion with insecticide resistance and a significant reduction in variability extending at least 20 kb downstream of the Cyp6g1 gene. The frequency of the Accord insertion differs significantly between East African (32-55%) and nonAfrican (85-100%) populations. This pattern is consistent with a selective sweep driving the Accord insertion close to fixation in nonAfrican populations as a result of the insecticide resistance phenotype it confers. This study confirms that hitchhiking mapping can be used to identify beneficial mutations in natural populations.     

Studies of effects of macromolecular crowding and confinement on protein folding and protein stability

In cells, proteins execute specific tasks in crowded environments; these environments influence their stability and dynamics. Similarly, for an enzyme molecule encapsulated in an inorganic cavity as in biosensors or biocatalysts, confinement or excluded volume plays an important role in its stability and dynamics. In this article we present results of our experimental and theoretical investigations of the confinement and macromolecular crowding effects on protein. On the experimental side we study the stability of encapsulated cytochrome c against unfolding induced by the presence of denaturants, such as urea. Results show that, as the pore size in which protein is trapped is reduced, protein shows higher stability against denaturant-induced unfolding. On the theoretical side, after reviewing our previous study of the confinement effects on the equilibrium and dynamic properties of protein using a minimalist (two-dimensional lattice, Monte Carlo, Brownian dynamics) model, we have extended the model so that the effects of macromolecular crowding on such properties can be studied. Our simulations show that both folding and unfolding times increase with the number of crowders in solution, however, the equilibrium constant is affected such that the equilibrium is shifted towards the folded state. Furthermore, our results show that, for a fixed number of crowders as the size of crowder (or excluded volume) increases, the average size of protein at equilibrium decreases.     

Plant Golgi-associated vesicles contain a novel alpha-actinin-like protein

By using Western blotting, immunofluorescence and immunogold labeling, a novel alpha-actinin-like protein was found in pollen and pollen tubes of Lilium davidii, a model system for cytoskeleton and Golgi apparatus study of plant. As measured by Western blotting, the molecular mass of the a-actinin-like protein was about 80 kDa. Under confocal laser scanning microscopy after immunofluorescence labeling, the distribution of the alpha-actinin-like protein appeared punctated in the cytoplasm of the pollen and pollen tubes. When double labeled, the protein was co-localized with Golgi 58K protein. In addition, some fraction of the alpha-actinin-like protein was found to co-distribute with F-actin bundles in the pollen tubes. Additional studies with immuno-gold labeling and transmission electron microscopy revealed that the alpha-actinin-like protein bound mainly to the membranes of Golgi-associated vesicles. When the pollen tubes were treated with Brefeldin A (BFA), the a-actinin-like proteins were dispersed into the cytoplasm, and the growth of pollen tubes was inhibited. After BFA was removed, the protein was reversibly recovered on the Golgi apparatus. These results suggest that the novel alpha-actinin-like protein is a BFA-sensitive protein on the membranes of Golgi-associated vesicles, and may participate in Golgi-associated vesicles budding and/or sorting, together with actin microfilaments.     

Comparison between effects of dantrolene and nifedipine on lipopolysaccharide-induced endotoxemia in the anesthetized rats

Intracellular calcium is an important mediator for regulating the cellular response in endotoxemia. In this study, we investigated the effects of dantrolene and nifedipine, two agents of reducing intracellular calcium levels, on bacterial endotoxin (lipopolysaccharide, LPS; 10 mg/kg i.v.)-induced production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) as well as hemodynamic changes in the anesthetized rat. Injection of LPS (i) induced biphasic changes of blood glucose and rectal temperature: an initial increased phase (<180 min after injection of LPS) followed by a decreased phase (at 240 or 360 min), (ii) caused a significant fall in mean arterial blood pressure from 119+/-3 mmHg (at time 0) to 73+/-67 mmHg (at 360 min) with a concomitant increase of heart rate, (iii) resulted in a substantial hyporeactivity to norepinephrine (NE) (1 microg/kg i.v.), (iv) increased plasma nitrate (an indicator of NO formation) in a time-dependent manner, and (v) induced bell-shape changes in plasma TNF-alpha levels which reached a peak at 60 min. Pretreatment of animals with dantrolene (1 mg/kg i.v. at 20 min prior to LPS) or nifedipine (20 microg/kg i.v. infusion for 20 min at 20 min prior to LPS) not only attenuated the delayed circulatory failure (e.g. delayed hypotension and vascular hyporeactivity to NE), but also prevented the overproduction of NO caused by LPS in the rat. However, the prevention of NO overproduction by dantrolene, but not by nifedipine, was associated with an inhibition of TNF-alpha production elicited by LPS. Thus, both dantrolene and nifedipine have beneficial hemodynamic effects, although through different mechanisms, in animals with endotoxic shock.     

Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation

In vitro evidence has suggested a change in the ability of tau bearing mutations associated with fronto-temporal dementia to promote microtubule assembly. We have used a cellular assay to quantitate the effect of both isoform differences and mutations on the physiological function of tau. Whilst all variants of tau bind to microtubules, microtubule extension is reduced in cells transfected with 3-relative to 4-repeat tau. Mutations reduce microtubule extension with the P301L mutation having a greater effect than the V337M mutation. The R406W mutation had a small effect on microtubule extension but, surprisingly, tau with this mutation was less phosphorylated in intact cells than the other variants.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

Human MPS1 kinase is required for mitotic arrest induced by the loss of CENP-E from kinetochores

We have determined that the previously identified dual-specificity protein kinase TTK is the human orthologue of the yeast MPS1 kinase. Yeast MPS1 (monopolar spindle) is required for spindle pole duplication and the spindle checkpoint. Consistent with the recently identified vertebrate MPS1 homologues, we found that hMPS1 is localized to centrosomes and kinetochores. In addition, hMPS1 is part of a growing list of kinetochore proteins that are localized to nuclear pores. hMPS1 is required by cells to arrest in mitosis in response to spindle defects and kinetochore defects resulting from the loss of the kinesin-like protein, CENP-E. The pattern of kinetochore localization of hMPS1 in CENP-E defective cells suggests that their interaction with the kinetochore is sensitive to microtubule occupancy rather than kinetochore tension. hMPS1 is required for MAD1, MAD2 but not hBUB1, hBUBR1 and hROD to bind to kinetochores. We localized the kinetochore targeting domain in hMPS1 and found that it can abrogate the mitotic checkpoint in a dominant negative manner. Last, hMPS1 was found to associate with the anaphase promoting complex, thus raising the possibility that its checkpoint functions extend beyond the kinetochore.     

Natural antimicrobial agent (reuterin) produced by lactobacillus reuteri for sanitization of biological tissues inoculated with pseudomonas aeruginosa

The study was done to evaluate the efficacy of using reuterin produced by Lactobacillus reuteri to sanitize biological tissues. The microorganism tested in the study was Pseudomonas aeruginosa, a common cause of nosocomial biomaterial-related infections. The inhibitory effect of reuterin on P. aeruginosa for an inoculated tissue was investigated at different conditions of concentration, temperature, and pH. Additionally, the cellular compatibility of the reuterin-sanitized tissue was evaluated. Glutaraldehyde was employed as a control. It was noted that the minimum inhibitory concentration (MIC, 33.0 +/- 2.9 ppm) and minimum bactericidal concentration (MBC, 50.0 +/- 0.0 ppm) values of reuterin for P. aeruginosa were significantly lower than their glutaraldehyde counterparts (MIC, 130.0 +/- 8.2 ppm and MBC, 180.0 +/- 18.3 ppm). This indicated that reuterin was more efficient than glutaraldehyde as an antimicrobial agent. The addition of reuterin on the inoculated tissue led to a reduced viability of P. aeruginosa. The reduction in the P. aeruginosa culture was more pronounced with increasing the concentration of reuterin (0-100 ppm). At increasing temperature (25-45 degrees C), there was an increasing effect of reuterin on its sanitization activity. However, it should be pointed out that the growth of P. aeruginosa in the nutrient broth was also significantly affected by temperature. The sanitization activity of reuterin was more evident with increasing the pH level (pH 6.5-8.5). The cytotoxicity of reuterin was significantly lower than that of glutaraldehyde. Additionally, the cellular compatibility of the reuterin-sanitized tissue was superior to its glutaraldehyde-sanitized counterpart.